Pure human fibroblast interferon (Hu IFN-beta) was used to raise antibodies in rabbits. The anti-IFN-beta immunoglobulins from rabbit serum were purified by IFN-beta affinity chromatography and used in the development of a sandwich type radioimmunoassay (RIA) for Hu IFN-beta. The RIA consisted of (1) incubating Hu IFN-beta with anti-IFN-beta immunoglobulins immobilized on Sepharose 4B beads and (2) incubating the resulting solid phase IFN-antibody complex with 125I-labelled anti-Hu IFN-beta immunoglobulins. The amount of 125I-labelled anti-Hu IFN-beta bound to the complex was linearly proportional to the amount of antiviral activity present in the complex from 0 unit to 500 units/ml of standard IFN-beta. One unit of antiviral activity was equivalent to about 40 cpm of 125I-anti-IFN immunoglobulins in the RIA. The results of the RIA were not influenced by the protein content of the IFN sample assayed and the degree of intra-assay variability was low; the coefficient of variability was 9.4% at 5 units/ml and 1.1% at 500 units/ml. Thus, compared with the traditional antiviral assay for IFN, this RIA was equally sensitive and more precise. In addition, it was highly specific for the Hu IFN-beta and took only 5 hours to complete.